Universal red blood cells, methods of preparing same, and uses thereof

Abstract

The present invention is directed to pegylated red blood cells comprising a polyethylene glycol (PEG) attached to a thiolated amino group on a membrane protein, and to compositions comprising the pegylated red blood cells. The invention also provides methods of preparing pegylated red blood cells comprising reacting red blood cells with a compound that produces a thiolated amino group on a red blood cell membrane protein, and reacting the thiolated red blood cell with a PEG. The invention further provides methods of treatment using pegylated red blood cells.

1. A method of preparing pegylated red blood cells comprising a polyethylene glycol (PEG) having a molecular weight of 5 K daltons and a polyethylene glycol (PEG) having a molecular weight of 20 K daltons, the method comprising the sequential steps of:
 (a) introducing a polyethylene glycol (PEG) having a molecular weight of 5 K daltons onto the red blood cells to prepare pegylated red blood cells comprising a polyethylene glycol (PEG) having a molecular weight of 5 K daltons, wherein either (1) the red blood cells are contacted with a chemical thiolating compound that produces a thiolated amino group on a membrane protein of the red blood cells and the polyethylene glycol (PEG) is attached to the thiolated amino group via a maleimide or (2) the polyethylene glycol (PEG) is a succinimidyl-propionate-activated (SPA)-polyethylene glycol (PEG); 
 (b) removing excess reagents; and 
 (c) introducing a polyethylene glycol (PEG) having a molecular weight of 20 K daltons onto the pegylated red blood cells comprising the polyethylene glycol (PEG) having a molecular weight of 5 K daltons to prepare pegylated red blood cells comprising a polyethylene glycol (PEG) having a molecular weight of 5 K daltons and a polyethylene glycol (PEG) having a molecular weight of 20 K daltons, wherein either (1) the red blood cells are contacted with a chemical thiolating compound that produces a thiolated amino group on a membrane protein of the red blood cells and the polyethylene glycol (PEG) is attached to the thiolated amino group via a maleimide or (2) the polyethylene glycol (PEG) is a succinimidyl-propionate-activated (SPA)-polyethylene glycol (PEG); 
 wherein the polyethylene glycol (PEG) is a methoxy PEG; and 
 wherein pegylation of the red blood cells masks a Rh antigen and one or more of A antigen and B antigen. 
 
     
     
       2. The method of  claim 1 , wherein step (a) comprises contacting the red blood cells with a chemical thiolating compound that produces a thiolated amino group on a membrane protein of the red blood cells and wherein the polyethylene glycol (PEG) is attached to the thiolated amino group via a maleimide. 
     
     
       3. The method of  claim 2 , wherein the chemical thiolating compound is iminothiolane. 
     
     
       4. The method of  claim 2 , wherein the chemical thiolating compound is dithio sulfo succinimidyl propinate (DTSSP). 
     
     
       5. The method of  claim 1 , wherein in step (a), the polyethylene glycol (PEG) is a succinimidyl-propionate-activated (SPA)-polyethylene glycol (PEG). 
     
     
       6. The method of  claim 1 , wherein step (c) comprises contacting the red blood cells with a chemical thiolating compound that produces a thiolated amino group on a membrane protein of the red blood cell and wherein the polyethylene glycol (PEG) is attached to the thiolated amino group via a maleimide. 
     
     
       7. The method of  claim 6 , wherein the chemical thiolating compound is iminothiolane. 
     
     
       8. The method of  claim 6 , wherein the chemical thiolating compound is dithio sulfo succinimidyl propinate (DTSSP). 
     
     
       9. The method of  claim 1  wherein in step (c), the polyethylene glycol (PEG) is a succinimidyl-propionate-activated (SPA)-polyethylene glycol (PEG). 
     
     
       10. The method of  claim 1 , wherein the polyethylene glycol (PEG) in step (a) is a maleimide methoxypolyethylene glycol. 
     
     
       11. The method of  claim 10 , wherein the maleimide methoxypolyethylene glycol is a maleimide phenyl methoxypolyethylene glycol. 
     
     
       12. The method of  claim 10 , wherein the maleimide methoxypolyethylene glycol is a maleimidophenyl carbamate of methoxypolyethylene glycol. 
     
     
       13. The method of  claim 1 , wherein the polyethylene glycol (PEG) in step (a) is a maleimide methoxypolyethylene glycol and wherein the polyethylene glycol (PEG) in step Cc) is a succinimidyl-propionate-activated (SPA)-polyethylene glycol (PEG). 
     
     
       14. The method of  claim 1 , wherein the Rh antigen is one or more of Rh(D), Rh(C), Rh(c), Rh(E) and Rh(e). 
     
     
       15. The method of  claim 1 , wherein pegylation of the red blood cells does not alter the oxygen affinity of the red blood cells by more than 20%. 
     
     
       16. The method of  claim 1 , wherein pegylation of the red blood cells does not alter the oxygen affinity of the red blood cells by more than 15%. 
     
     
       17. The method of  claim 1 , wherein the method is carried out at a temperature between about room temperature and about 4° C. 
     
     
       18. The method of  claim 1 , wherein the method is carried out at a pH of about pH 7.4-9.2. 
     
     
       19. The method of  claim 1 , wherein the method is carried out for about 2 hours. 
     
     
       20. A pegylated red blood cell comprising a polyethylene glycol (PEG) having a molecular weight of 5 K daltons and a polyethylene glycol (PEG) having a molecular weight of 20 K daltons produced by the method of  claim 1 . 
     
     
       21. A composition comprising the pegylated red blood cell of  claim 20  and a pharmaceutically acceptable carrier.